Method of selecting soybeans with enhanced bioactivity and compositions for reducing cancer cell viability

Abstract

The invention provides a method that uses enzyme-treatment of whole soybeans or partially defatted soybeans to select soybeans with improved bioactivity or bioactivities. The invention further provides a soybean plant and seed with a non-transgenic mutation conferring enhanced bioactivity as an hydrolysate when compared to hydrolysate from other seeds, for instance in a cell-based assay, including reduced cancer cell viability; increased LDL receptor activity; reduced lipid accumulation; increased adiponectin expression; decreased FAS and LPL expression; reduced production of NO and PGE 2 , and expression of iNOS and COX-2; higher antioxidant activity; promotion of growth of bifidobacteria; and inhibiting the growth of pathogenic bacteria; for instance when compared to other seeds tested as hydrolysates. The invention also provides soybean plants for use in producing seeds that have an overall improved bioactivity compared to other seeds as hydrolysates by combining effects on several bioactivity assays in a health index. The invention also provides products derived from, and parts of, these plants and uses thereof. Methods for producing such plants are also provided, as well as methods for standardizing or assuring quality control of soybean products with enhanced bioactivity for humans and animals.

1 . A method of selecting soybeans for human food with improved or standardized bioactivity associated with human health, wherein
 a) ground soybeans are heat-treated in water and digested with one or more protease(s); and   b) the digest or extract of the digest is included in a bioactivity assay.   
     
     
         2 . The method of  claim 1 , wherein the one or more protease(s) are selected from the group consisting of pepsin, pancreatin, and alcalase. 
     
     
         3 . The method of  claim 1 , wherein the soybeans are dehulled soybeans, edamame or roasted soybeans. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein
 i) the ground soybeans are defatted prior to digestion;   ii) the heat-treatment prior to digestion is performed for from about 5 minutes to about 3 hours;   iii) the heat-treatment prior to digestion is performed at about 50° C. to about 85° C.:   iv) the heat-treatment prior to digestion is performed at about 50° C. for about 3 hours; or   v) the heat-treatment prior to digestion is performed at about 85° C. for about 5 minutes.   
     
     
         7 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein other enzymes are included such as lipase, alpha-amylase, trypsin and or chymotrypsin. 
     
     
         11 . The method of  claim 1 , wherein
 i) the digest is freeze-dried;   ii) the bioactivity assay comprises a chemical assay;   iii) the bioactivity assay comprises a chemical assay and antioxidant activity is determined.   
     
     
         12 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the heat treatment prior to digestion is performed for from about 5 minutes to about 3 hours and wherein
 i) bile acid binding is determined;   ii) a negative bioactivity is determined;   iii) a negative bioactivity is determined using the Ames assay;   iv) the bioactivity assay is a cell-based bioassay;   v) L1210 leukemia mouse line cells are used as a model to test soybeans for anticancer bioactivity;   vi) adipocyte cells are used as a model to test reduced lipid accumulation activity;   vii) macrophages are used as a model to test anti-inflammatory activity;   viii) the expression of mRNA for adiponectin is measured in adipocyte cells;   ix) expression of mRNA for LDL receptors and enzymes such as fatty acid synthetase and HMG CoA reductase is measured in liver cell lines;   x) expression of mRNA for enzymes such as fatty acid synthetase and lipoprotein lipase is measured in cell lines; or   xi) expression of mRNA for CCK, GLP-1, and PYY is determined in an enteroendocrine cell line.   
     
     
         15 .- 24 . (canceled) 
     
     
         25 . The method of  claim 14  wherein the enteroendocrine cell line comprises STC-1 cells. 
     
     
         26 . A soybean plant capable of producing seeds with enhanced bioactivity related to human and animal health. 
     
     
         27 . A seed of the plant of  claim 26 , with enhanced bioactivity related to human and animal health wherein
 a) the bioactivity comprises reduced cancer cell viability;   b) the bioactivity comprises reduced cancer cell viability and wherein the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content;   c) the bioactivity comprises increased LDL receptor activity;   d) the bioactivity comprises increased inhibition of lipid accumulation;   e) the bioactivity is increased adiponectin expression; or   f) the bioactivity is increased adiponectin expression and the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content.   
     
     
         28 .- 29 . (canceled) 
     
     
         30 . The soybean seed of  claim 27 , wherein the bioactivity comprises increased LDL receptor activity and wherein the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content. 
     
     
         31 . The soybean seed of  claim 30 , wherein the Kunitz trypsin inhibitor content is less than 2% of the total protein content. 
     
     
         32 . The soybean seed of  claim 27 , wherein the bioactivity comprises increased inhibition of lipid accumulation and wherein the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content. 
     
     
         33 . (canceled) 
     
     
         34 . The soybean seed of  claim 32 , wherein the Kunitz inhibitor content is less than 2% of the total protein. 
     
     
         35 . The soybean seed of  claim 27 , wherein the bioactivity is reduced LPL or FAS expression. 
     
     
         36 . The soybean seed of  claim 35 , wherein the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content. 
     
     
         37 . The soybean seed of  claim 27 , wherein the bioactivity is increased antioxidant capacity. 
     
     
         38 . The soybean seed of  claim 37 , wherein the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content. 
     
     
         39 .- 40 . (canceled) 
     
     
         41 . A method of producing a soybean plant with enhanced bioactivity for human and animal health, comprising:
 a) screening at least two soybean seeds for an enhanced bioactivity for human or animal health;   b) selecting a seed having the enhanced bioactivity for human or animal health; and   c) growing a first plant from the selected seed.   
     
     
         42 . The method of  claim 41 , further comprising the steps of:
 d) crossing the first plant to a second soybean plant to form a population; and   e) selecting and growing at least one seed from the first or second plant having enhanced bioactivity for human or animal health.   
     
     
         43 . A soybean protein product with enhanced bioactivity for human or animal health. 
     
     
         44 . The product of  claim 43 , wherein
 a) the β-conglycinin content is greater than 27% of total protein content and the glycinin content is less than 30% of total protein content;   b) the product is further defined as comprising an omega-3 fatty acid or a conjugated linoleic acid;   c) the product is further defined as comprising an omega 3-fatty acid and a conjugated linoleic acid; or   d) the product is selected from the group consisting of: extruded soybean, soybean meal, soyflour, defatted soyflour, soymilk, spray-dried soymilk, soy protein concentrate, texturized soy protein, hydrolyzed soy protein, soy protein isolates, and spray-dried tofu.   
     
     
         45 .- 47 . (canceled) 
     
     
         48 . A foodstuff made with the soybean protein product of  claim 44 , selected from the group consisting of: a pet food, a beverage, an infused food, a sauce, a soup, coffee creamer, a cookie, an emulsifying agent, bread, candy, an instant milk drink, gravy, noodles, soynut butter, soy coffee, roasted soybeans, a cracker, soymilk, tofu, tempeh, baked soybeans, a bakery ingredient, a bar, beverage powder, breakfast cereal, fruit juice, syrup, dessert, icing and fillings, a soft frozen product, a confection, and an intermediate food. 
     
     
         49 . A method of inhibiting inflammation, pain or itching, comprising: applying to a subject a topical composition comprising β-conglycinin or a protease-treated preparation of β-conglycinin. 
     
     
         50 . The method of  claim 49  wherein the composition comprises:
 a) a protease-treated preparation made from soybean seed wherein at least 27% of the total protein in the soybean seed comprises beta-conglycinins and less than 30% of the total protein in the soybean seed comprises glycinins; and 
 b) a cosmetically acceptable topical carrier. 
 
     
     
         51 . A method of preventing or inhibiting inflammation and/or associated pain, comprising: consuming a food or drink made from soybean seed wherein at least 27% of the total protein in the soybean seed comprises beta-conglycinins and less than 30% of the total protein in the soybean seed comprises glycinins. 
     
     
         52 . The method of  claim 51  where the food is selected from the group consisting of: a liquid form of edamame; a powder form of edamame; roasted soybeans; soymilk; soy kefir; soy yogurt; soy flour; soy protein concentrate; soy protein isolate; and β-conglycinin isolate. 
     
     
         53 . The method of  claim 52  where the soymilk is whole bean soymilk. 
     
     
         54 . The method of  claim 53  where the soymilk is treated for over one hour with a protease. 
     
     
         55 . The method of  claim 54  where the protease is alcalase. 
     
     
         56 . The method of  claim 55  wherein consumption follows physical exercise or participation in a sporting competition. 
     
     
         57 . The method of  claim 53  wherein the β-conglycinin or β-conglycinin peptides is (are) administered to a subject in the amount of about 1 gram to about 50 grams per day. 
     
     
         58 . A topical composition comprising β-conglycinin or a protease-treated preparation of β-conglycinin. 
     
     
         59 . The composition of  claim 58  wherein the composition comprises:
 a) a protease-treated preparation made from soybean seed wherein at least 27% of the total protein in the soybean seed comprises β-conglycinins and less than 30% of the total protein in the soybean seed comprises glycinins; and 
 b) a cosmetically acceptable topical carrier. 
 
     
     
         60 . The composition of  claim 58 , comprising β-conglycinin, wherein greater than 33% of the total soy protein or peptides originate from β-conglycinins. 
     
     
         61 . A method of selecting soybeans and another plant protein source for human food with improved or standardized bioactivity associated with human health, wherein
 a) ground soybeans and at least one additional whole plant component source or sources are heat-treated;   b) the heat treated preparation of step (a) is digested with one or more proteases to form a digest or an extract of the digest; and   c) the digest or extract of the digest is included in a bioactivity assay.   
     
     
         62 . The method of  claim 61  where the plant component source or sources are selected from the group consisting of: sunflower seeds, flax seeds, a nut, and a vegetable. 
     
     
         63 . The method of  claim 62 , wherein the vegetable is spinach, broccoli, tomatoes, peppers, lettuce, purple carrot, bean, or cucumber. 
     
     
         64 . A method of enhancing the growth of beneficial microflora in an animal gut comprising: consuming a food or drink comprising the product of  claim 43 . 
     
     
         65 . The method of  claim 64 , wherein the animal is a human.